Covalent attachment of multi-ubiquitin chains to cellular proteins represents a potent degradative signaling mechanism. Multi-ubiquitinated proteins are recognized by a specific protease; the target protein is degraded, while ubiquitin is regenerated. Successive ubiquitins in multi-ubiquitin chains are linked by isopeptide bonds involving the side chain of Lys-48 and the ubiquitin C-terminus. The mammalian ubiquitin- conjugating enzyme E2-25K has a high specific activity in synthesis of multi-ubiquitin chains from isolated ubiquitin, and has a high (amino acid) sequence similarity to a subfamily of yeast E2s which play an essential role in protein degradation. Comprehensive studies are proposed to address the hypothesis that E2-25K plays a predominant role in multi-ubiquitination and protein degradation in mammalian cells. A reverse genetic method (antisense RNA) will be used to interfere with the expression of E2-25K; the effects on protein turnover rates, and on the level of free multi-ubiquitin chains within cells, will be determined. Deletional analysis of E2-25K suggests that its C-terminus is a determinant of conjugative specificity; we will further test this hypothesis by evaluating the conjugative specificity of a chimeric protein in which the E2-25K C-terminus is fused to the core of a different E2. These results will be interpreted in the light of detailed structural information obtained by X-ray crystallographic analysis of the E2, and of an analog of the catalytic E2-ubiquitin thiol ester intermediate. The results of the proposed studies may identify E2-25K as a ubiquitin-conjugating enzyme which functions globally in protein turnover in mammalian cells, and moreover have potential to provide the first direct link (in any system) between E2 structure, conjugative specificity, and biological function. Other experiments will address post-conjugative aspects of the structure-function relationship in ubiquitin itself. Multi-ubiquitin chains bearing reactive groups at defined positions on the chain surface will be used in cross-linking studies with the ubiquitin conjugate protease. The results will address the global mode of binding of chains to the protease, and should reveal which components of this multi-subunit enzyme are involved in conjugate recognition.